X-ray crystallography of membrane proteins, many of which are important drug targets, is a challenging and critically important field of research. Unfortunately, it lags significantly behind x-ray diffraction studies of cytosolic proteins. There are two specific technical problems that account for this: lack of efficient expression systems for membrane proteins (especially those of eukaryotic origin) and difficulties in the preparation of single crystals suitable for x-ray diffraction. The crystals that are obtained using current techniques are often of poor quality and typically diffract in the range of 3.0 - 5.0 A, yielding relatively imprecise atomic models of the target proteins. Studies conducted by the Principal Investigator established that rational surface mutagenesis of soluble proteins can be a powerful method allowing for the preparation of crystals of proteins otherwise recalcitrant to structural studies, and in some cases to obtain novel crystal forms diffracting to much higher resolution than wild-type crystals. The approach, based on the concept of surface entropy reduction (SER), is the first successful attempt designed to rationally induce crystallization by engineering epitopes that mediate crystal contacts and consequently lead to nucleation and growth. To date, a number of crystals of new proteins have been obtained using this method. We now propose to use similar principles to conduct a feasibility study of improving the quality of membrane protein crystals by engineering better crystal contacts. To that end, we will use two proteins as model systems: the E. coil mechanosensitive and voltage modulated channel (MscS), and the E. coli aquaporin AqpZ. These proteins can be overexpressed with relatively high yields, making them particularly suitable for our purposes. The MscS published structure was limited to 3.9 A resolution, while none of the several crystals forms of AqpZ yielded X-ray data to a resolution warranting structural investigation. Our pilot study, within the high risk/high impact framework of an R21 proposal, will aim to establish the feasibility of a longer-term research into rational surface mutagenesis of membrane proteins for high-resolution crystallographic studies.